Method for analyzing the ability of a substance to cross a bilayer membrane

Patent metadata

Field	Value
Publication number	US-2019242914-A1
Application number	US-201716315452-A
Country	US
Kind code	A1
Filing date	Jul 4, 2017
Priority date	Jul 4, 2016
Publication date	Aug 8, 2019
Grant date	—

Abstract

The present invention relates to a method to analyse the ability of a substance (3) to cross a bilayer membrane comprising the following steps: (i) providing in a hydrophobic medium (1) a first droplet D1 of an aqueous solution AS1, wherein the droplet D1 is surrounded by a monolayer of amphiphilic molecules (2), (ii) providing in the said hydrophobic medium (1) a second droplet D2 of an aqueous solution AS2 containing the substance (3) to be analysed, wherein the droplet D2 is surrounded by a monolayer of amphiphilic molecules (2), (iii) putting the first droplet D1 and the second droplet D2 into contact so as to form a bilayer of amphiphilic molecules (2) in the contact area and to have a contact angle 2θ between the two droplets D1 and D2 comprised between 10° and 180°, (iv) adding to the hydrophobic medium (1) a solvent capable of separating the droplets D1 and D2 put into contact, (v) collecting the droplet D1, and (vi) analysing the content of the droplet D1 in order to determine the presence or not of the substance (3).

First claim

1 . A method to analyse the ability of a substance to cross a bilayer membrane comprising the following steps:
(i) providing in a hydrophobic medium a first droplet D 1 of an aqueous solution AS 1 , wherein the droplet D 1 is surrounded by a monolayer of amphiphilic molecules, (ii) providing in the said hydrophobic medium a second droplet D 2 of an aqueous solution AS 2 containing the substance to be analysed, wherein the droplet D 2 is surrounded by a monolayer of amphiphilic molecules, (iii) putting the first droplet D 1 and the second droplet D 2 into contact so as to form a bilayer of amphiphilic molecules in the contact area and to have a contact angle 2θ between the two droplets D 1 and D 2 comprised between 90° and 180°, (iv) adding to the hydrophobic medium a solvent capable of separating the droplets D 1 and D 2 put into contact, (v) collecting the droplet D 1 , and (vi) analysing the content of the droplet D 1 in order to determine the presence or not of the substance.

2 . The method according to claim 1 , wherein the hydrophobic medium is an oil, triglycerides, a silicone oil, a high molecular weight hydrocarbon or a mixture thereof; optionally in admixture with a solvent selected from a halogenated hydrocarbon, an alcohol, an ether, a fatty acid, a diglyceride, an ester, a low molecular weight hydrocarbon and a mixture thereof.

3 . The method according to claim 1 , wherein the amphiphilic molecules are phospholipids, glycolipids, monoglycerides, cholesterol, or a mixture thereof.

4 . The method according to claim 3 , wherein the amphiphilic molecules are phospholipids.

5 . The method according to claim 4 , wherein the amphiphilic molecules are selected from dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylglycerol (DOPG), and mixtures thereof.

6 . The method according to claim 1 , wherein the aqueous solutions AS 1 and AS 2 contain a buffering agent.

7 . The method according to claim 1 , wherein the first and second droplets D 1 and D 2 have a diameter comprised between 0.5 μm and 1000 μm.

8 . The method according to claim 1 , wherein step (i) is performed by:
providing the aqueous solution AS 1 , providing the hydrophobic medium further containing the amphiphilic molecules, and forming a droplet of the aqueous solution AS 1 in the hydrophobic medium which contains the amphiphilic molecules.

9 . The method according to claim 1 , wherein step (ii) is performed by:
providing the aqueous solution AS 2 containing the substance, providing the hydrophobic medium further containing the amphiphilic molecules, and forming a droplet of the aqueous solution AS 2 containing the substance in the hydrophobic medium which contains the amphiphilic molecules.

10 . The method according to claim 1 , wherein the contact angle 2θ is comprised between 120° and 180°.

11 . The method according to claim 1 , wherein the droplets D 1 and D 2 are maintained into contact in step (iii) during at least 1 min.

12 . The method according to claim 1 , wherein the solvent capable of separating the droplets D 1 and D 2 put into contact is selected from a halogenated hydrocarbon; an alcohol; an ether; a fatty acid; a diglyceride; an ester; a low molecular weight hydrocarbon; and a mixture thereof.

13 . The method according to claim 1 , wherein step (vi) is performed by means of fluorescence imaging, HPLC or mass spectrometry.

14 . The method according to claim 1 , used in high-throughput.

15 . The method according to claim 1 , performed by means of a microfluidic analysis system comprising:
a microfluidic device comprising:
a first microfluidic channel comprising one inlet and one outlet, through which alternate droplets D 1 and D 2 can flow in the hydrophobic medium from the inlet to the outlet of the first microfluidic channel and can be put into contact,
a second microfluidic channel comprising one inlet and one outlet, through which the droplets D 1 and D 2 can flow in the hydrophobic medium from the inlet to the outlet of the second microfluidic channel and the droplets D 1 and D 2 put into contact can be separated, the inlet of the second microfluidic channel being connected to the outlet of the first microfluidic channel,
at least two outlets connected to the outlet of the second microfluidic channel, wherein:
the first outlet of the microfluidic device is further connected to a first receiver container intended to receive the droplets D 1 to be analysed, and
the second outlet of the microfluidic device is further connected to a second receiver container intended to receive the droplets D 2 ,
at least five inlets:
wherein four inlets of the microfluidic device are connected to the inlet of the first microfluidic channel,
the first two inlets being further connected respectively to a reservoir intended to contain the aqueous solution AS 1 and to a reservoir intended to contain the hydrophobic medium and the amphiphilic molecules, and
the last two inlets being further connected respectively to a reservoir intended to contain the aqueous solution AS 2 and the substance and to a reservoir intended to contain the hydrophobic medium and the amphiphilic molecules, and
wherein one inlet of the microfluidic device is connected to the inlet of the second microfluidic channel and is further connected to a reservoir intended to contain the solvent capable of separating the droplets D 1 and D 2 put into contact,
at least four reservoirs adapted for containing respectively (i) the aqueous solution AS 1 , (ii) the aqueous solution AS 2 and the substance, (iii) the hydrophobic medium and the amphiphilic molecules, and (iv) the solvent capable of separating the droplets D 1 and D 2 put into contact, at least two receiver containers adapted for receiving respectively (i) the droplets D 1 to be analyzed, and (ii) the droplets D 2 , and an analyzing device to analyze the content of droplets D 1 .

16 . The method according to claim 4 , wherein the amphiphilic molecules are phosphatidylcholines (PC), phosphatidylethanolamines (PE), dioleoylphosphatidylglycerol (PG), dioleoylphosphatidylserine (DOPS), or a mixture thereof.

17 . The method according to claim 7 , wherein the first and second droplets D 1 and D 2 have a diameter comprised between 50 μm and 200 μm.

18 . The method according to claim 11 , wherein the droplets D 1 and D 2 are maintained into contact in step (iii) between 10 and 30 min.

19 . The method according to claim 12 , wherein the solvent capable of separating the droplets D 1 and D 2 put into contact is selected from chloroform, dichloromethane, butanol, diethyl ether, 1,4-dioxane, oleic acid, glyceryl dioctanoate, glyceryl dioleate, ethyl acetate, isopropyl acetate, pentane, cyclopentane, hexane, cyclohexane, benzene, toluene, and a mixture thereof.

20 . The method according to claim 19 , wherein the solvent capable of separating the droplets D 1 and D 2 put into contact is chloroform.

Assignees

• Paris Sciences Lettres Quartier Latin
• Univ Paris Diderot Paris 7
• Centre Nat Rech Scient
• Univ Sorbonne

Inventors

• Thiam Abdou Rachid

Classifications

• G01N33/92 Primary

  involving lipids, e.g. cholesterol {, lipoproteins, or their receptors (steroid hormones G01N33/743)} · CPC title

• B01L2300/087

  Multiple sequential chambers · CPC title

• B01L3/502784

  specially adapted for droplet or plug flow, e.g. digital microfluidics · CPC title

• B01L2300/165

  Specific details about hydrophobic, oleophobic surfaces · CPC title

• B01L2300/047

  Additional chamber, reservoir · CPC title